The purpose of this project is to develop kinetic tools for studying enzyme mechanisms, and to apply them to representative enzymes. During the coming grant period the major emphasis will be on the use of C-13, N-15 and O-18 isotope effects. 1) Isotope effects will be measured at C-1of ribose and N-1 of nicotinamide in the NAD substrate, and at the carbonyl carbon and oxygen of the acetyl-peptide substrate in the reaction catalyzed by Sir2. Sir2 is a member of the silent regulator 2 family of protein deacetylases (sirtuins) which are involved in a host of cellular processes such as gene silencing, apoptosis, cell cycle regulation, fatty acid metabolism, and life span extension. Our isotope effect studies of this system will help unravel this mechanism and should help in the design of sirtuin inhibitors. 2) Isotope effects will be determined at the carbon and oxygen of the carboxyl group of nicotinic acid adenine dinucleotide and in ammonia in the reaction catalyzed by NAD synthetase (M. tuberculosis). This synthetase is an important drug target for the treatment of turberculosis. The characterization of the transition state structure for this reaction will aid in the design of a potent inhibitor.